The present invention relates to a process for detecting the presence or absence of a conserved, nucleotide sequence of a virus related to human T cell leukemia virus-types I and II (HTLVI and II). This invention also relates to a kit for such detection having primers and a labeled hybridization probe.
A family of T cell tropic retroviruses, known as human T cell leukemia viruses (HTLV), is known to be involved in the pathogenesis of certain T cell neoplasms. Currently, there exist three known types of HTLV. The first, type I (HTLVI), is an oncovirus that has been linked to a human adult T-cell leukemia-lymphoma (ATLL) that is found in Japan, the Caribbean region, and Africa. The second, type II (HTLVII), is an oncovirus that has been isolated from two patients having a T-cell variant of hairy cell leukemia. See M. Popovic et al., Science, 224:497-500 (1984) and Rosenblatt, J. D. et al., New Eng. J. Of Med., August, 1986. The third, type III (HTLVIII), is a lentivirus and is the aetiologic agent responsible for acquired immune deficiency syndrome (AIDS), a transmissible disorder of the cellular immune system resulting in frequently fatal opportunistic infections.
The current immunodiagnostic tests to identify sera with antibodies to the HTLV-associated virus(es) such as AIDS (see U.S. Pat. No. 4,520,113 to Gallo et al.) are being used in blood banks to eliminate potentially infectious blood. See also WO 86/01834 published Mar. 27, 1986 (University of California) for retroviral polypeptides useful in preparing monoclonal antibodies to detect retroviruses in the HTLV family Because the viruses may reside as a DNA copy without producing significant quantities of viral particles, a direct immunological approach to detect HTLVI and II-related viruses may prove unsuccessful in a significant fraction of persistently infected asymptomatic individuals. Because the number of virus particles in the infected tissues and blood may be few (due to viral quiescence), direct detection of viral particles or RNA/DNA may be difficult, if not impossible, without co-culturing the infected cells with a permissive T cell line.
U.S. Pat. No. 4,683,202 describes a process for amplifying nucleic acid sequences to facilitate detection thereof, as by using a labeled RNA or DNA hybridization probe. In this process primers are used to obtain primer extension products which are used as templates to synthesize additional complementary strands in the presence of nucleotides. The above-mentioned patent application also describes a technique whereby after a probe is hybridized to the desired sequence, a restriction enzyme is added to cleave the hybrid at a site within the desired sequence, and the restriction digest is then analyzed for labeled fragments. U.S. Pat. No. 4,683,194 and Saiki et al., Biotechnology, 3:1008-1012 (1985) describe this latter technique in greater detail. Both patent applications illustrate use of the process for detecting genetic diseases such as sickle cell anemia and .beta.-thalassemia. These methods and the process for amplifying nucleic acid sequences are also disclosed in Saiki et al., Science, 230, 1350-1354 (1985), the disclosure of which is incorporated herein by reference.
A review article by Landry et al., Clin. Lab. Med. (1985) 5, 513-529 describes the field of nucleic acid hybridization as applied to virus detection. W086/01535 published Mar. 13, 1986 and EP 173,529 published Mar. 5, 1986 disclose molecular cloning of HTLVIII and use of the clone as a probe to detect AIDS. Further, EP patent publication 173,339, published Mar. 5, 1986, discloses a genetic analysis using a DNA probe to detect infections by foreign microbes. EP 185,444, published June 25, 1986, discloses a recombinant peptide for use as a probe to detect the HTLVIII virus in cell lysates. Oncor Inc. announced in September, 1986 that it has developed a radioactive blood test to detect the AIDS virus. Finally, copending U.S. application Ser. No. 935,581, filed Nov. 26, 1986, now abandoned, which is a continuation-in-part of Ser. No. 818,127 filed Jan. 10, 1986, now abandoned disclosed a method for detecting AIDS viruses using the amplification procedure described above together with a hybridization probe. This application is related to U.S. Ser. No. 934,955, now abandoned, file Nov. 26, 1986, entitled "Detection of Viruses by Amplification and Hybridization."
Use of a hybridization probe to detect the oncoviruses HTLVI and II may allow identification of those individuals who are persistently infected but are not producing virus or individuals who are antibody negative but culture positive, and to detect infected cells without the need to culture the virus. Increasing the viral nucleic acid copy number of the virus by amplification will facilitate the identification of viral nucleic aicd in infected individuals.